The present invention is directed to microfluidic devices having multiple microcoil nuclear magnetic resonance (NMR) detectors and, more particularly to microfluidic devices having improved microcoil NMR detectors for capillary-scale, high resolution NMR spectroscopy probes capable of enhanced sample processing functionality.
Nuclear magnetic resonance spectroscopy, or NMR, is a powerful and commonly used method for analysis of the chemical structure of molecules. NMR provides spectral information as a function of the electronic environment of the molecule and is nondestructive to the sample. In addition, reaction rates, coupling constants, bond-lengths, and two- and three-dimensional structure can be obtained with this technique.
Systems for biochemical, chemical, and molecular analysis can be miniaturized as capillary-based systems or substrate-based, i.e., micro-scale, systems with multifunctional capabilities including, for example, chemical, optical, fluidic, electronic, acoustic, and/or mechanical functionality. Miniaturization of these systems offers several advantages, including increased complexity, functionality, and efficiency. Devices can be fabricated from diverse materials including, for example, plastics, polymers, metals, silicon, ceramics, paper, and composites of these and other materials. Mesoscale sample preparation devices for providing microscale test samples are described in U.S. Pat. No. 5,928,880 to Wilding et al. Devices for analyzing a fluid sample, comprising a solid substrate microfabricated to define at least one sample inlet port and a mesoscale flow channel extending from the inlet port within the substrate for transport of a fluid sample are described in U.S. Pat. No. 5,304,487. Currently known miniaturized fluid-handling and detection devices have not met all of the needs of industry.
NMR is one of the most information-rich forms of biochemical, chemical, and molecular detection and analysis, and remains highly utilized in a wide range of health-related industries, including pharmaceutical research and drug discovery. One of the fundamental limitations of NMR for these and other applications involves sample throughput. When compared to other forms of detection (e.g., mass spectrometry), the amount of sample required by NMR is generally orders of magnitude greater, and correspondingly the mass limits of detection are generally orders of magnitude poorer. Conventional NMR spectrometers typically use relatively large RF coils (mm to cm size) and samples in the ml volume range, and significant performance advantages are achieved using NMR microcoils when examining very small samples. Prior to such development of microcoil NMR and NMR flowprobes, NMR remained a test tube-based analytical technique requiring milliliters of sample and often requiring data acquisition times ranging from 10 min. to several hours for informative spectra with sufficient signal to noise ratio (xe2x80x9cS/Nxe2x80x9d).
NMR microcoils are known to those skilled in the art and are shown, for example, in U.S. Pat. No. 5,654,636 to Sweedler et al., and in U.S. Pat. No. 5,684,401 to Peck et al., and in U.S. Pat. No. 6,097,188 to Sweedler et al., all three of which patents are incorporated herein by reference in their entireties for all purposes. A solenoid microcoil detection cell formed from a fused silica capillary wrapped with copper wire has been used for static measurements of sucrose, arginine and other simple compounds. Wu et al. (1994a), J. Am. Chem. Soc. 116:7929-7930; Olson et at. (1995), Science 270:1967-1970, Peck (1995) J. Magn. Reson. 108(B) 114124. Coil diameter has been further reduced by the use of conventional micro-electronic techniques in which planar gold or aluminum R.F. coils having a diameter ranging from 10-200 xcexcm were etched in silicon dioxide using standard photolithography. Peck 1994 IEEE Trans Biomed Eng 41(7) 706-709, Stocker 1997 IEEE Trans Biomed Eng 44(11)1122-1127, Magin 1997 IEEE Spectrum 34 51-61, which are also incorporated herein by reference in its entirety for all purposes. In Stocker et al. a microcoil was fabricated on a gallium arsenide substrate with an inner diameter of 60 xcexcm, an outer diameter of 200 xcexcm, trace width of 10 xcexcm, trace spacing of 10 xcexcm, and trace height of Sum. At 5.9T (250 MHz) in 1H-NMR micro spectroscopy experiments using a spectral width of 1 kHz, 4096 sampled data points, and a recovery delay of 1 s, a SNR of 25 (per acquisition) and a spectral line width of less than 2 Hz were obtained from a sample of water.
Miniature total analysis systems (xcexc-TAS) are discussed in Integrating Microfluidic Systems And NUR Spectroscopyxe2x80x94Preliminary Results, Trumbull et al, Solid-State Sensor and Actuator Workshop, pp. 101-05 (1998), Magin 1997 IEEE Spectrum 34 51-61, and Trumbull 2000 47(1)1-6 incorporated herein by reference in its entirety for all purposes. These groups constructed chip-based capillary electrophoresis (CE) devices equipped with an integrated planar radio frequency detector coil used for nuclear magnetic resonance spectroscopy (NN R). Separations were accomplished in the devices, but satisfactory NMR spectra could only be obtained from samples of high concentration. Two prototype CE-NMR devices are presented that represent complete microanalytical instruments. Further, xe2x80x9cThe first system, Trident, was designed to be a proof-of-concept fluidic-NMR device to gauge the effectiveness of integrated, single-turn planar NMR coils. The channel network was formed by solvent bonding a photopatterned polyimide coating (on a glass slide) with a cover-glass coated with a thin layer of polyimide. Holes were previously drilled ultrasonically in the glass slide to provide access. A lift-off process was used to create a 1 mm diameter, single-turn coil on the outer surface of the cover glass. The metal was formed from 3 evaporated layers: Cr/Cu/Cr with respective thicknesses of 150, 9700, and 150 angstroms for improved susceptibility matching. The resistance of the coil, pad to pad, was measured to be 5.90. Acrylic wells were then placed over the drilled holes and bonded with epoxy. The second device type created, SpinCollector shown in FIG. 1 blowups, was made from etched glass channels using methods developed from (D. J. Harrison and N. Chiem, xe2x80x9cImmunoassay Flow Systems On-Chip,xe2x80x9d TRF, pp. 5-8., 1996). Annealed Pyrex glass wafers (1 mm thick) were etched in HF and HNO3 to a depth of 20 xcexcm through a Cr/Au mask. Access holes were drilled ultrasonically and, the mask was stripped. The wafers were then cleaned in a 1% HF bath for 1 minute with ultrasonic agitation to remove any loose glass particles. After thorough cleaning, the wafers were thermally bonded to unprocessed pieces forming closed channels. A 5 mm diameter, single-turn coil was then formed through lift-off on the undrilled cover-glass slide over the disk-shaped reservoir, and glass wells were attached using epoxy. The Trumbull et al. device integrated multiple chemical processing steps and the means of analyzing their results on the same miniaturized system. Specifically, Trumbull et al. coupled chip-based capillary electrophoresis (CE) with nuclear magnetic resonance spectroscopy (NMR) in a xcexc-TAS system.
Capillary-based liquid chromatography and microcoil NMR have compatible flow rates and sample volume requirements. Thus, for example, the combination of the Waters CapLC(trademark) available from Waters Corporation (Milford, Mass., USA) and the MRM CapNMR(trademark) flow probe available from MRM Corporation (Savoy, Ill.), a division of Protasis Corporation (Marlborough, Mass., USA) provides excellent separation capability in addition to UV-VIS and NMR detection for mass-limited samples. The Waters CapLC(trademark) has published flow rates from 0.02 xcexcL/minute to 40 xcexcL/minute. A typical CapLC on-column flow rate is 5 xcexcL/min, the autosampler-injected analyte volume is 0.1 xcexcL or more, and accurate flow rates are achieved through capillary of typically 50 xcexcm inner diameter. The NMR flow cell has a typical total volume of 5 xcexcL with a microcoil observe volume of 1 xcexcL. A typical injected sample amount for CapLC-xcexcNMR analysis is a few xcexcg (nmol) or less.
Capillary scale systems also are shown in U.S. Pat. No. 6,194,900, the entire disclosure of which is incorporated herein by reference for all purposes. In such systems, a capillary-based analyte extraction chamber is connected to an NMR flow site, such as by being positioned as an operation site along a capillary channel extending to the NMR flow cell.
Small volume flow probes are shown, for example, by Haner et al. in Small Volume Plow Probe for Automated Direct-Injection NAM Analysis: Design and Performance, J. Magn. Reson., 143, 69-78 (2000), the entire disclosures of which is incorporated herein by reference for all purposes. Specifically, Harter et al show a tubeless NMR probe employing an enlarged sample chamber or flowcell. In Haner et al., a 600 MHz, indirect detection NMR flow probe with a 120 xcexcL active volume is evaluated in two configurations: first as a stand-alone small volume probe for the analysis of static nonflowing solutions, and second as a component in an integrated liquids-handling system used for high-throughput NMR analysis. Key advantages of the flowprobe include high molar sensitivity, ease of use in an automation setup, and superior reproducibility of magnetic field homogeneity, which enables the practical implementation of 1 D T2-edited analysis of protein-ligand interactions. Microcoil-based micro-NMR spectroscopy is disclosed in U.S. Pat. Nos. 5,654,636, 5,684,441, and 6,097,188, the entire disclosures of all of which are incorporated herein by reference for all purposes. Sample amounts can now range as small as several hundred microliters for conventional flowprobes to smaller than 1 xcexcL for microcoil-based capillary-scale flowprobes. Acquisition times typically range from minutes to hours. The most expensive and technologically limiting component of the NMR system is the superconducting magnet. Although significant financial and technical investment has been made in the development of elaborate mechanical (robotic-controlled) sample changers and, more recently, automated flow injection systems for repetitive and continuous sample throughput, the magnet remains today a dedicated component in which only sequential, one-at-a-time analysis of samples is carried out.
NMR is one of the few analytical methods in which parallel data acquisition has not been applied to increase sample processing functionality, such as the number of samples that can be tested in a given time. At least some of the difficulties in accomplishing this objective are intrinsically related to the hardware involved in NMR data acquisition.
Recent academic results have shown that some of the limitations of NMR processing can be overcome by the use of multiple microcoil detectors in a wide-bore magnet. Proposed designs for incorporating multiple solonoidal microcoils into a single probe head are presented by Li et al. in Multiple Solenoidal Microcoil Probes for High-Sensitivity, High-Throughput Nuclear Magnetic Resonance Spectroscopy, Anal. Chem., 71, 4815-4820, 1999. A dual channel probe for simultaneous acquisition of NMR data from multiple samples is shown by Fisher et al. in NMR Probe for the Simultaneous Acquisition of Multiple Samples, J. Magn. Reson., 138, 160-163 (1999). Such devices, however, have not been commercially implemented and have not been shown to be commercially viable. In addition, higher numbers of multiple microcoil detectors are needed that are compatible also with narrow bore magnets, since narrow bore magnets are predominant in industrial settings. There is also both need for and benefit of microcoil NMR probes having enhanced sample processing functionality.
Accordingly, it is an object of the present invention to provide multi-microcoil NMR microfluidic devices having enhanced sample processing functionality. It is a particular object of the invention to provide improved microcoil NMR detectors for capillary-scale, high resolution NMR spectroscopy probes that can be adapted in accordance with certain preferred embodiments for use in large or small bore magnets and that are capable of enhanced sample processing functionality. Given the benefit of this disclosure, additional objects and features of the invention, or of certain preferred embodiments of the invention, will be apparent to those skilled in the art, that is, those skilled in this area of technology.
In accordance with a first aspect, an NMR system comprises an NMR probe comprising multiple NMR detection sites. Each of the multiple NMR detection sites comprises a sample holding void and an associated NMR microcoil. The NMR system further comprises a controllable fluid router operative to direct fluid sample to the multiple NMR detection sites. In accordance with certain preferred embodiments, the multiple NMR sites are integrated in a probe module further disclosed below. In accordance with certain preferred embodiments, each of the NMR detection sites is in a capillary-scale fluid channel in the module. In accordance with certain preferred embodiments, each of the NMR detection sites is in a micro-scale fluid channel in the module. In accordance with certain preferred embodiments, the controllable fluid router is operative in response to an electrical input signal, especially to direct fluid sample to any selected ones of the NMR detection sites. In accordance with certain preferred embodiments, the NMR system further comprises a controller unit in communication with the router and operative to generate the input signal to the router. In certain embodiments the NMR system controller unit is operative to receive information from any of the multiple NMR detection sites and to generate the input signal to the router based at least in part on that information. In accordance with certain preferred embodiments, the NMR system further comprises a data processing unit which may be remote from the probe module or integral therewith. The data processing unit can provide the aforesaid input signal to the controllable router.
In accordance with a second aspect, an NMR probe comprises multiple NMR detection sites as disclosed above, each comprising a sample holding void and an associated NMR microcoil, and a controllable fluid router operative to direct fluid sample to the multiple NMR detection sites.
In accordance with another aspect, an NMR xe2x80x9csmart probexe2x80x9d comprises multiple NMR detection sites, each comprising a sample holding void and an associated NMR microcoil, a controllable fluid router operative in response to an electrical input signal to direct fluid sample to the multiple NMR detection sites, and a controller unit in communication with the router and operative to generate the input signal to the router.
In accordance with another aspect, an NMR probe module is provided, e.g., a module suitable to be interchangeably (i.e., removeably and optionally reuseable) installed in certain preferred embodiments of the NMR probes disclosed above. Such probe modules comprise at least one fluid inlet port operative to receive a fluid sample, a fluid pathway comprising multiple channels in fluid communication with the inlet port, for the transport of fluid sample to be tested, multiple NMR detection sites, each in fluid communication with at least one of the multiple channels and each comprising a sample holding void and an associated NMR microcoil, and a controllable fluid router operative to direct fluid sample in the module to at least a selected one of the multiple channels.
The multiple NMR sites optionally can be optimized for different nuclear species and/or for 1 or 2 dimensional NMR study, e.g., the sites can be optimized similarly or differently, using different materials, such as fused silica and PEEK, fused silica and polytetrofluoroethylene and/or other suitable materials known to those skilled in those skilled in the art.
In accordance with another aspect, an NMR probe module comprises at least one fluid inlet port, operative to receive a fluid sample, a fluid pathway comprising multiple channels in fluid communication with the at least one fluid inlet port, for the transport of fluid sample to be tested, and multiple NMR detection cells, each in fluid communication with a corresponding one of the multiple channels. Each of the multiple NMR detection cells comprises an associated NMR microcoil and an enlarged void for holding a fluid sample. In certain preferred embodiments, the NMR probe module further comprises a controllable fluid router as disclosed above.